The invention relates to the removal of electronic noise from mass spectra which are scanned as single spectra and added together to give a sum spectrum.
Many types of mass spectrometer obtain single spectra in rapid succession. These contain the signals of only a few ions and are therefore of poor quality in regard to the reproducibility of the signal intensities for each ion species in the mass spectrum. These spectra, sometimes scanned at very high frequencies of several kilohertz, are then immediately added up in the computer system of the mass spectrometer to form a sum spectrum in order to obtain a usable spectrum for the ion species of different masses with signals which have less fluctuation. The addition is also used to increase the measurement dynamics since very fast digitizers with rates in the GHz range have data-bus widths of only 8 bits.
At this point, it would be appropriate to describe a few very different examples of these types of mass spectrometer:
Time-of-Flight mass spectrometer with ionization by Matrix-Assisted Laser Desorption and Ionization (MALDI-TOF). In this case, typically, 50-200 and in some instruments even 1,000 spectra are added. These are scanned at a rate of 10-100 spectra per second and with a scanning width of up to 200,000 measurement points per spectrum. The digitizing rate is approximately one to four GHz with a conversion width of 8 bits. 5-100 ms are available for adding the spectra, depending on the scanning rate -i.e. 25-500 ns per measurement point. In most cases, the spectra are transferred to a computer after each single spectrum is scanned and not processed further until they have arrived.
Time-of-flight mass spectrometers with Orthogonal Time-of-Flight (OTOF) where an analog-to-digital converter is used. In this case, 1,000-5,000 spectra are added. These are scanned at a rate of 20,000 spectra per second. Each spectrum contains 25,000 measurement points; the digitizing rate is approximately 500 MHz with a data bus width of 8 bits. The addition takes place in digitizing transient recorders which have been specially developed for this task. The spectra are scanned directly one after the other; thus, only 2 ns are available for each addition. The transient recorders have been specially developed for low background noise, which has to be lower than one count of a digitized converter. In spite of this, switching peaks are still present. Even if these only amount to one bit each and only appear occasionally, if they always appear in the same place, they easily add up to form pseudopeaks which have nothing to do with the genuine ion peaks.
Ion-Trap Mass Spectrometers (ITMS) usually operate with the addition of only 5, and in borderline cases up to 200, spectra, depending on the analytical task. The spectra are scanned at the rate of 5 to 10 spectra per second and each spectrum contains up to 50,000 measurement points. The digitizing rate is 300 kHz and uses a data bus width of 12 to 16 bits; the electronic noise amounts to a few counts of the digitized measurement value. Large numbers of spectra are required, especially for the analysis of large biomolecules with ionization by static nanospray, since there are only a few ions in the part of the measurement range which can be evaluated. The electron-spray ionization (ESI) method which is usually used causes the ions to spread across many charge states; there are therefore very large numbers of ion species giving mass signals with different mass-to-charge ratios; only occasionally an ion signal adds to such a mass signal during subsequent spectrum scans.
Each single mass spectrum usually contains electronic noise along with the ion signals. At high conversion bus widths from 12 to 16 bits, the electronic noise usually amounts to a few counts of the digital converter. At smaller conversion bus widths of 8 bits, the noise is less and the background signal usually amounts to the same count but in this case, both the conversion rates and the numbers of single spectra which have to be summed are very large.
The ions can be normal ions which add up to produce a mass signal (also referred to as a mass peak) in the sum spectrum or scatter ions which, by avoiding the clean, mass spectrometric ion separation, fall on the detector at some point in time to produce an ion signal. When the spectra are added, the scatter ions do not produce a mass peak to indicate the presence of ion species of a certain mass-to-charge ratio but add up to form a broad band of background noise which cannot be separated from the summed electronic noise.
In all of the mass spectrometers listed above, secondary electronic multipliers (SEV) are used for measuring the ion beams. These basically can be adjusted so that a single ion gives a signal which stands out from the electronic noise. When these spectra are summed, the ion signals are added together, but so is the electronic noise. The zero point of the amplifier is usually adjusted so that the center line of the noise signal is somewhat above the zero line and therefore it is possible to check on the spectrum that none of the useful signal is cut off. Accordingly, during the addition process, the center line of the noise increases as does the noise itself; the center line increases linearly with the number of spectra and the noise increases with the root of the number of spectra.
One means which is occasionally used to suppress the electronic noise consists of suppressing the center line of the noise to below the zero line of the analog-to-digital converter (ADC) by applying a slight negative bias voltage to the preamplifier (the amplifier before the conversion of the analog value into a digital value). In this way, the electronic noise of each single spectrum is cut off, but with a similar amount of the useful signal. However, since the center line of the noise over the single spectrum can move into the positive or negative area over the mass range, this method cannot always be applied without cutting off large portions of the useful signal. Apart from this, the method removes any control over the drift of the zero line, which means, for example, that the center-line drift caused by temperature effects can no longer be detected and corrected.
The technique which has been used until now involves smoothing the background noise and removing the background from the sum spectrum alone. By so doing, mass peaks consisting of only a few ions are regularly lost since they no longer stand out from the noise. The technique derives from an era when computers were still too slow to process single spectra in any way whatsoever before summation.
The basic idea of the invention is to eliminate the electronic noise from the single spectra (and no longer from the sum spectrum) by using very fast computers and computer methods since in the single spectra it is still possible to distinguish between electronic noise and ion signalsxe2x80x94even those of ions appearing individually. Since the summation of the spectra to form a sum spectrum regularly takes place in real time (if only because of the enormous quantities of memory which would otherwise be needed), there is very little time available. However, with skilful programming, the very fast signal processors which are available today can perform this task even for very high spectral-scanning frequencies.
With a moderate amount of processor time, a noise band either side of the center line of the noise is defined and all signal values which do not exceed the noise band are not added to the sum spectrum; the value of the center line is subtracted from all the signal values which do exceed the noise band before they are added to the sum spectrum. In this case, the width of the noise band is selected so that it is smaller than the signal height of a single ion. It is expedient for the center line of the noise to be calculated as a sliding average value over a predeterminable number of measurements for this purpose.
In a simpler and faster embodiment of the invention, during the summation process of the single spectra, only those measured values which exceed a certain threshold value are added to the sum spectrum. For spectra in which an accurate quantitative evaluation is not essential, the center value for the noise does not need to be subtracted. However, even without subtracting the average noise, quantitative evaluation is possible after appropriate calibration.
In another embodiment of the invention, which requires more processor time, the width and the position of the noise band can be adjusted dynamically. The position of the noise band can be controlled by the position of the sliding average value. If, for example, less than 30% (adjustable) of the measured values are located in the interval for the sliding average value, then the width of the noise band can be increased automatically by an adjustable width step. It has been found from a few types of mass spectrometry that the electronic noise in the spectrum increases as the ion masses increase in size; in other types of mass spectrometry, an increased proportion of noise has been observed in certain areas of the spectrum. For a new single spectrum, the noise band is then reset to the initial value.
The noise band can also be spread asymmetrically, depending on the number of signal values which exceed the band, upwards or downwards.
The initial value reset can also be controlled dynamically, for example, by using the result of the sliding average value calculation at the beginning of the spectrum last scanned as the initial value and by using the initial standard deviation of the last spectrum for establishing the width of the noise band.
In another embodiment of the invention, the sliding average is also used to regulate the zero-line adjustment by setting the bias voltage of the pre-amplifier to specified values.
The result of this measure, which sounds simple but is not so simple technically, is surprising since the resulting spectra are of a quality and degree of freedom from noise previously unknown. It has been found that, with well-designed mass spectrometers which have been designed to keep the proportion of vagabond scattered ions small, spectra are obtained which not only have no electronic noise by are also practically free of background noise caused by scattered ions. In large numbers of single spectra, ions which were previously regarded as scattered ions in the single spectra are summed to provide sensible mass signals.
Finally, this measure certainly makes it possible to distinguish between vagabond scattered ions and ions which, when added, form mass peaks. This invention can be used to improve the mass spectrometer in regard to suppressing the vagabond scattered ions.
Particularly in ion traps, eliminating the electronic noise also results in a significant improvement in the control of the optimum number of ions. When filling the ion traps with substances at very low concentration which produce only very weak ion beams, it is possible to approach the overdriving limit for the amplifier more closely. This significantly improves the limits of detection for these substances.